Chimeric binding peptide library screening method

ABSTRACT

There is described a method of isolating nucleotide sequences encoding target peptides from DNA libraries using DNA binding proteins to link the peptide to the sequence which encodes it. DNA libraries are prepared from cells encoding the protein of interest, or from synthetic DNA, and inserted into, or adjacent to, a DNA binding protein in an expression vector to create a chimeric fusion protein. Incorporation of the vector DNA into a carrier package, during expression of the chimeric fusion protein, results in the production of a peptide display carrier package (PDCP) displaying the DNA-bound fusion protein on the external surface of the carrier package. Employment of affinity purification techniques results in the PDCP particles containing sequences encoding the desired peptide to be selected and the desired nucleotide sequences obtained therefrom.

The present invention relates generally to methods for screeningnucleotide libraries for sequences that encode peptides of interest.

Isolating an unknown gene which encodes a desired peptide from arecombinant DNA library can be a difficult task. The use ofhybridisation probes may facilitate the process, but their use isgenerally dependent on knowing at least a portion of the sequence of thegene which encodes the protein. When the sequence is not known, DNAlibraries can be expressed in an expression vector, and antibodies havebeen used to screen for plaques or colonies displaying the desiredprotein antigen. This procedure has been useful in screening smalllibraries, but rarely occurring sequences which are represented in lessthan about 1 in 10⁵ clones (as is the case with rarely occurring cDNAmolecules or synthetic peptides) can be easily missed, making screeninglibraries larger than 10⁶ clones at best laborious and difficult.Methods designed to address the isolation of rarely occurring sequencesby screening libraries of 10⁶ clones have been developed and includephage display methods and LacI fusion phage display, discussed in moredetail below.

Phage display methods. Members of DNA libraries which are fused to theN-terminal end of filamentous bacteriophage pIII and pVIII coat proteinshave been expressed from an expression vector resulting in the displayof foreign peptides on the surface of the phage particle with the DNAencoding the fusion protein packaged in the phage particle (Smith G. P.,1985, Science 228: 1315-1317). The expression vector can be thebacteriophage genome itself, or a phagemid vector, into which abacteriophage coat protein has been cloned. In the latter case, the hostbacterium, containing the phagemid vector, must be co-infected withautonomously replicating bacteriophage, termed helper phage, to providethe full complement of proteins necessary to produce mature phageparticles. The helper phage normally has a genetic defect in the originof replication which results in the preferential packaging of thephagemid genome. Expression of the fusion protein following helper phageinfection, allows incorporation of both fusion protein and wild typecoat protein into the phage particle during assembly. Libraries offusion proteins incorporated into phage, can then be selected forbinding members against targets of interest (ligands). Bound phage canthen be allowed to reinfect Escherichia coli (E. coli) bacteria and thenamplified and the selection repeated, resulting in the enrichment ofbinding members (Parmley, S. F., & Smith, G. P. 1988., Gene 73: 305-318;Barrett R. W. et al., 1992, Analytical Biochemistry 204: 357-364Williamson et al., Proc. Natl. Acad. Sci. USA, 90: 4141-4145; Marks etal., 1991, J. Mol. Biol. 222: 581-597).

Several publications describe this method. For example, U.S. Pat. No.5,403,484 describes production of a chimeric protein formed from theviral coat protein and the peptide of interest. In this method at leasta functional portion of a viral coat protein is required to causedisplay of the chimeric protein or a processed form thereof on the outersurface of the virus. In addition, U.S. Pat. No. 5,571,698 describes amethod for obtaining a nucleic acid encoding a binding protein, a keycomponent of which comprises preparing a population of amplifiablegenetic packages which have a genetically determined outer surfaceprotein, to cause the display of the potential binding domain on theouter surface of the genetic package. The genetic packages are selectedfrom the group consisting of cells, spores and viruses. For example whenthe genetic package is a bacterial cell, the outer surface transportsignal is derived from a bacterial outer surface protein, and when thegenetic package is a filamentous bacteriophage, the outer surfacetransport signal is provided by the gene pIII (minor coat protein) orpVIII (major coat protein) of the filamentous phage.

WO-A-92/01047 and WO-A-92/20791 describe methods for producingmultimeric specific binding pairs, by expressing a first polypeptidechain fused to a viral coat protein, such as the gene pIII protein, of asecreted replicable genetic display package (RGDP) which displays apolypeptide at the surface of the package, and expressing a secondpolypeptide chain of the multimer, and allowing the two chains to cometogether as part of the RGDP.

LacI fusion plasmid display. This method is based on the DNA bindingability of the lac repressor. Libraries of random peptides are fused tothe lacI repressor protein, normally to the C-terminal end, throughexpression from a plasmid vector carrying the fusion gene. Linkage ofthe LacI-peptide fusion to its encoding DNA occurs via the lacOsequences-on the plasmid, forming a stable peptide-LacI-peptide complex.These complexes are released from their host bacteria by cell lysis, andpeptides of interest isolated by affinity purification on an immobilisedtarget. The plasmids thus isolated can then be reintroduced into E. coliby electroporation to amplify the selected population for additionalrounds of screening (Cull, M. G. et al. 1992. Proc. Natl. Acad. Sci.U.S.A. 89:1865-1869).

U.S. Pat. No. 5,498,530 describes a method for constructing a library ofrandom peptides fused to a DNA binding protein in appropriate host cellsand culturing the host cells under conditions suitable for expression ofthe fusion proteins intra-cellularly, in the cytoplasm of the hostcells. This method also teaches that the random peptide is located atthe carboxy terminus of the fusion protein and that the fusionprotein-DNA complex is released from the host cell by cell lysis. Nomethod is described for the protection of the DNA from degradation oncereleased from the lysed cell. Several DNA binding proteins are claimedbut no examples are shown except lacI.

There remains a need for methods of constructing peptide libraries inaddition to the methods described above. For instance, the above methodsdo not permit production of secreted peptides with a free carboxyterminus. The present invention describes an alternative method forisolating peptides of interest from libraries and has significantadvantages over the prior art methods.

In general terms, the present invention provides a method for screeninga nucleotide library (usually a DNA library) for a nucleotide sequencewhich encodes a target peptide of interest. The method involvesphysically linking each peptide to a polynucleotide including thespecific nucleotide sequence encoding that peptide. Linkage of a peptideto its encoding nucleotide sequence is achieved via linkage of thepeptide to a nucleotide binding domain. A bifunctional chimeric proteinwith a nucleotide binding domain and a library member or target peptide(preferably with a function of interest) is thus obtained. The peptideof interest is bound to the polynucleotide encoding that peptide via thenucleotide binding domain of the chimeric protein.

The polynucleotide-chimeric protein complex is then incorporated withina peptide display carrier package (PDCP), protecting the polynucleotidefrom subsequent degradation, while displaying the target peptide portionon the outer surface of the peptide display carrier package (PDCP).

Thus, in one aspect, the present invention provides a peptide displaycarrier package (PDCP), said package comprising apolynucleotide-chimeric protein complex wherein the chimeric protein hasa nucleotide binding portion and a target peptide portion, wherein saidpolynucleotide comprises a nucleotide sequence motif which isspecifically bound by said nucleotide binding portion, and wherein atleast the chimeric protein encoding portion of the polynucleotide notbound by the nucleotide binding portion of the chimeric protein isprotected.

In one embodiment the polynucleotide is protected by a protein whichbinds non-specifically to naked polynucleotide. Examples include viralcoat proteins, many of which are well-known in the art. Where the chosenviral coat protein requires an initiation sequence to commence generalbinding to the polynucleotide, this will be provided on thepolynucleotide at appropriate location(s). A preferred coat protein iscoat protein from a bacteriophage, especially M13.

Generally, the nucleic binding portion of the chimeric protein isselected for its specificity for the nucleotide sequence motif presentin the recombinant polynucleotide encoding the chimeric protein.

Optionally, the nucleotide sequence motif may be an integral part of theprotein encoding region of the polynucleotide. Alternatively, and moreusually, the motif may be present in a non-coding region of thepolynucleotide. For the purposes of this invention, all that is requiredis for the motif to be located on the polynucleotide such that thenucleotide binding portion of the chimeric protein is able to recogniseand bind to it. Desirably the polynucleotide-chimeric protein complexhas a dissociation constant of at least one hour.

Optionally, the recombinant polynucleotide may comprise two or morenucleotide sequence motifs, each of which will be bound by a chimericprotein molecule. Preferably, the motifs are positioned along the lengthof the polynucleotide to avoid steric hindrance between the boundchimeric proteins.

Preferably, the nucleotide sequence motif is not affected by thepresence of additional nucleotide sequence (e.g., encoding sequence) atits 5′ and/or 3′ ends. Thus the chimeric fusion protein may include atarget peptide portion at its N terminal end, at its C terminal end ormay include two target peptide portions (which may be the same ordifferent) at each end of the nucleotide binding portion, ie at both theN and C terminal ends of the chimeric protein. For example one targetpeptide may be an antibody of known specificity and the other peptidemay be a peptide of potential interest.

Desirably the target peptide portion of the chimeric protein isdisplayed externally on the peptide display carrier package, and is thusavailable for detection, reaction and/or binding.

In more detail the PDCP may be composed two distinct elements:

-   -   a. A polynucleotide-chimeric protein complex. This links the        displayed target peptide portion to the polynucleotide encoding        that peptide portion through a specific polynucleotide binding        portion. The nucleotide sequence encoding the chimeric protein,        and the specific nucleotide sequence motif recognised by the        nucleotide binding portion of the chimeric protein must be        present on a segment of polynucleotide which can be incorporated        into the PDCP; and    -   b. A protective coat. This may be supplied by a replicable        carrier or helper package capable of independent existence.        Alternatively, a coat protein could be encoded by the        recombinant polynucleotide of the invention. The protective coat        for the polynucleotide-chimeric protein complex may be composed        of a biological material such as protein or lipid, but the        protective coat is not required for linking the target peptide        to the polynucleotide encoding that peptide. The protective coat        must allow the display of the target peptide portion of the        chimeric protein on its outer surface. The carrier or helper        package may also provide the mechanism for releasing the intact        PDCP from host cells when so required. By way of example, when a        bacteriophage is the replicable carrier package, a protein coat        of the bacteriophage surrounds the polynucleotide-chimeric        protein complex to form the PDCP, which is then extruded from        the host bacterial cell.

The invention described herein demonstrates that peptides fused to anucleotide binding domain can be displayed externally, even through abacteriophage carrier package protein coat, while still bound to thepolynucleotide encoding the displayed peptide.

The present invention also provides a recombinant polynucleotidecomprising a nucleotide sequence encoding a chimeric protein having anucleotide binding portion operably linked to a target peptide portion,wherein said polynucleotide includes a specific nucleotide sequencemotif which is bound by the nucleotide binding portion of said chimericprotein and further encoding a non-sequence-specific nucleotide bindingprotein.

Desirably, the recombinant polynucleotide is a recombinant expressionsystem, able to express the chimeric protein when placed in a suitableenvironment, for example a compatible host cell. After its expression,the chimeric protein binds to the specific nucleotide sequence (motif)present in the polynucleotide comprising the nucleotide sequenceencoding the chimeric protein.

Optionally there may be a linker sequence located between the nucleotidesequence encoding the nucleotide binding portion and the polynucleotideinserted into the restriction enzyme site of the construct.

Desirably the nucleotide binding portion is a DNA binding domain of anoestrogen or progesterone receptor, or a functional equivalent thereof.Examples of sequences encoding such nucleotide binding portions are setout in SEQ ID Nos 11 and 13.

The term “expression system” is used herein to refer to a geneticsequence which includes a protein-encoding region and is operably linkedto all of the genetic signals necessary to achieve expression of thatregion. Optionally, the expression system may also include regulatoryelements, such as a promoter or enhancer to increase transcriptionand/or translation of the protein encoding region or to provide controlover expression. The regulatory elements may be located upstream ordownstream of the protein encoding region or within the protein encodingregion itself. Where two or more distinct protein encoding regions arepresent these may use common regulatory element(s) or have separateregulatory element(s).

Generally, the recombinant polynucleotide described above will be DNA.Where the expression system is based upon an M13 vector, usually thepolynucleotide binding portion of the expressed chimeric portion will besingle-stranded DNA. However, other vector systems may be used and thenucleotide binding portion may be selected to bind preferentially todouble-stranded DNA or to double or single-stranded RNA, as convenient.

Additionally the present invention provides a vector containing such arecombinant expression system and host cells transformed with such arecombinant expression system (optionally in the form of a vector).

Whilst the recombinant polynucleotide described above forms an importantpart of the present invention, we are also concerned with the ability toscreen large (e.g. of at least 10⁵ members, for example 10⁶ or even 10⁷members) libraries of genetic material. One of the prime considerationstherefore is the provision of a recombinant genetic construct into whicheach member of said library can individually be incorporated to form therecombinant polynucleotide described above and to express the chimericprotein thereby encoded (the target peptide of which is encoded by thenucleotide library member incorporated into the construct).

Thus viewed in a further aspect the present invention provides a geneticconstruct or set of genetic constructs comprising a polynucleotidehaving a sequence which includes:

-   i) a sequence encoding a nucleotide binding portion able to    recognise and bind to a specific sequence motif;-   ii) the sequence motif recognised and bound by the nucleotide    binding portion encoded by (i);-   iii) a restriction enzyme site which permits insertion of a    polynucleotide, said site being designed to operably link said    polynucleotide to the sequence encoding the nucleotide binding    portion so that expression of the operably linked polynucleotide    sequences yields a chimeric protein; and-   iv) a sequence encoding a nucleotide binding protein which binds    non-specifically to naked polynucleotide.

Optionally there may be a linker sequence located between the nucleotidesequence encoding the nucleotide binding portion and the sequence of thepolynucleotide from the library inserted into the restriction enzymesite of the construct.

Desirably the nucleotide binding portion is a DNA binding domain of anoestrogen or progesterone receptor, or a functional equivalent thereof.Examples of sequences encoding such nucleotide binding portions are setout in SEQ ID Nos 11 and 13.

Suitable genetic constructs according to the invention include pDM12,pDM14 and pDM16, deposited at NCIMB on 28 Aug. 1998 under Nos NCIMB40970, NCIMB 40971 and NCIMB 40972 respectively.

It is envisaged that a conventionally produced genetic library may beexposed to the genetic construct(s) described above. Thus, eachindividual member of the genetic library will be separately incorporatedinto the genetic construct and the library will be present in the formof a library of recombinant polynucleotides (as described above),usually in the form of vectors, each recombinant polynucleotideincluding as library member.

Thus, in a further aspect, the present invention provides a library ofrecombinant polynucleotides (as defined above) wherein eachpolynucleotide includes a polynucleotide obtained from a genetic libraryand which encodes the target peptide portion of the chimeric proteinexpressed by the recombinant polynucleotide.

Optionally, the chimeric protein may further include a linker sequencelocated between the nucleotide binding portion and the target peptideportion. The linker sequence will reduce steric interference between thetwo portions of the protein. Desirably the linker sequence exhibits adegree of flexibility.

Also disclosed are methods for constructing and screening libraries ofPDCP particles, displaying many different peptides, allowing theisolation and identification of particular peptides by means of affinitytechniques relying on the binding activity of the peptide of interest.The resulting polynucleotide sequences can therefore be more readilyidentified, re-cloned and expressed.

A method of constructing a genetic library, said method comprising:

-   a) constructing multiple copies of a recombinant vector comprising a    polynucleotide sequence which encodes a nucleotide binding portion    able to recognise and bind to a specific sequence motif (and    optionally also including the specific sequence motif);-   b) operably linking each said vector to a polynucleotide encoding a    target polypeptide, such that expression of said operably linked    vector results in expression of a chimeric protein comprising said    target peptide and said nucleotide binding portions; wherein said    multiple copies of said operably linked vectors collectively express    a library of target peptide portions;-   c) transforming host cells with the vectors of step b);-   d) culturing the host cells of step c) under conditions suitable for    expression of said chimeric protein;-   e) providing a recombinant polynucleotide comprising the nucleotide    sequence motif specifically recognised by the nucleotide binding    portion and exposing this polynucleotide to the chimeric protein of    step d) to yield a polynucleotide-chimeric protein complex; and-   f) causing production of a non-sequence-specific moiety able to bind    to the non-protected portion of the polynucleotide encoding the    chimeric protein to form a peptide display carrier package.

The present invention further provides a method of screening a geneticlibrary, said method comprising:

-   a) exposing the polynucleotide members of said library to multiple    copies of a genetic construct comprising a nucleotide sequence    encoding a nucleotide binding portion able to recognise and bind to    a specific sequence motif, under conditions suitable for the    polynucleotides of said library each to be individually ligated into    one copy of said genetic construct, to create a library of    recombinant polynucleotides;-   b) exposing said recombinant polynucleotides to a population of host    cells, under conditions suitable for transformation of said host    cells by said recombinant polynucleotides;-   c) selecting for transformed host cells;-   d) exposing said transformed host cells to conditions suitable for    expression of said recombinant polynucleotide to yield a chimeric    protein; and-   e) providing a recombinant polynucleotide comprising the nucleotide    sequence motif specifically recognised by the nucleotide binding    portion and exposing this polynucleotide to the chimeric protein of    step d) to yield a polynucleotide-chimeric protein complex;-   f) protecting any exposed portions of the polynucleotide in the    complex of step e) to form a peptide display carrier package; and-   g) screening said peptide display carrier package to select only    those packages displaying a target peptide portion having the    characteristics required.

Desirably in step a) the genetic construct is pDM12, pDM14 or pDM16.

Desirably in step f) the peptide display package carrier is extrudedfrom the transformed host cell without lysis of the host cell.

Generally the transformed host cells will be plated out or otherwisedivided into single colonies following transformation and prior toexpression of the chimeric protein.

The screening step g) described above may look for a particular targetpeptide either on the basis of function (e.g. enzymic activity) orstructure (e.g. binding to a specific antibody). Once the peptidedisplay carrier package is observed to include a target peptide with thedesired characteristics, the polynucleotide portion thereof (which ofcourse encodes the chimeric protein itself) can be amplified, cloned andotherwise manipulated using standard genetic engineering techniques.

The current invention differs from the prior art teaching of theprevious disclosures U.S. Pat. Nos. 5,403,484 and 5,571,698, as theinvention does not require outer surface transport signals, orfunctional portions of viral coat proteins, to enable the display ofchimeric binding proteins on the outer surface of the viral particle orgenetic package.

The current invention also differs from the teaching of WO-A-92/01047and WO-A-92/20791, as no component of a secreted replicable geneticdisplay package, or viral coat protein is required, to enable display ofthe target peptide on the outer surface of the viral particle.

The current invention differs from the teaching of U.S. Pat. No.5,498,530, as it enables the display of chimeric proteins, linked to thepolynucleotide encoding the chimeric protein, extra-cellularly, not inthe cytoplasm of a host cell. In the current invention the chimericproteins are presented on the outer surface of a peptide display carrierpackage (PDCP) which protects the DNA encoding the chimeric protein, anddoes not require cell lysis to obtain access to the chimeric protein-DNAcomplex. Finally, the current invention does not rely upon the lacI DNAbinding protein to form the chimeric protein-DNA complex.

In one embodiment of the invention, the nucleotide binding portion ofthe chimeric protein comprises a DNA binding domain from one or more ofthe nuclear steroid receptor family of proteins, or a functionalequivalent of such a domain. Particular examples include (but are notlimited to) a DNA binding domain of the oestrogen receptor or theprogesterone receptor, or functional equivalents thereof. These domainscan recognise specific DNA sequences, termed hormone response elements(HRE), which can be bound as both double and single-stranded DNA. TheDNA binding domain of such nuclear steroid receptor proteins ispreferred.

The oestrogen receptor is especially referred to below by way ofexample, for convenience since:

-   (a) The oestrogen receptor is a large multifunctional polypeptide of    595 amino acids which functions in the cytoplasm and nucleus of    eukaryotic cells (Green et al., 1986, Science 231: 1150-1154). A    minimal high affinity DNA binding domain (DBD) has been defined    between amino acids 176 and 282 (Mader et al., 1993, Nucleic Acids    Res. 21: 1125-1132). The functioning of this domain (i.e. DNA    binding) is not inhibited by the presence of non-DNA binding domains    at both the N and C terminal ends of this domain, in the full length    protein.-   (b) The oestrogen receptor DNA binding domain fragment (amino acids    176-282) has been expressed in E. coli and shown to bind to the    specific double stranded-DNA oestrogen receptor target HRE    nucleotide sequence, as a dimer with a similar affinity (0.5 nM) to    the parent molecule (Murdoch et al. 1990, Biochemistry 29:    8377-8385; Mader et al., 1993, Nucleic Acids Research 21:    1125-1132). DBD dimerization on the surface of the PDCP should    result in two peptides displayed per particle. This bivalent display    can aid in the isolation of low affinity peptides and peptides that    are required to form a bivalent conformation in order to bind to a    particular target, or activate a target receptor. The oestrogen    receptor is capable of binding to its 38 base pair target HRE    sequence, consensus sequence:

SEQ ID No 77 1) 5′-TCAGGTCAGAGTGACCTGAGCTAAAATAACACATTCAG-3′(“minus strand”), and SEQ ID No 78 2)3′-AGTCCAGTCTCACTGGACTCGATTTTATTGTGTAAGTC-5′ (“plus strand”),with high affinity and specificity, under the salt and pH conditionsnormally required for selection of binding peptides. Moreover, bindingaffinity is increased 60-fold for the single-stranded coding, or “plus”,strand (i.e. SEQ ID No 78) of the HRE nucleotide sequence over thedouble stranded form of the specific target nucleotide sequence (Pealeet al. 1988, Proc. Natl. Acad. Sci. USA 85: 1038-1042; Lannigan &Notides, 1989, Proc. Natl. Acad. Sci. USA 86: 863-867).

In an embodiment of the invention where the DNA binding component of thepeptide display carrier package is the oestrogen receptor, thenucleotide (DNA) binding portion-contains a minimum sequence of aminoacids 176-282 of the oestrogen receptor protein. In addition, theconsensus oestrogen receptor target HRE sequence is cloned in such a waythat if single stranded DNA can be produced-then the coding, or “plus”,strand of the oestrogen receptor HRE nucleotide sequence is incorporatedinto single-stranded DNA. An example of a vector suitable for thispurpose is pUC119 (see Viera et al., Methods in Enzymology, Vol 153,pages 3-11, 1987).

In a preferred embodiment of the invention a peptide display carrierpackage (PDCP) can be assembled when a bacterial host cell istransformed with a bacteriophage vector, which vector comprises arecombinant polynucleotide as described above. The expression vectorwill also comprise the specific nucleotide motif that can be bound bythe nucleotide binding portion of the chimeric protein. Expression ofrecombinant polynucleotide results in the production of the chimericprotein which comprises the target peptide and the nucleotide bindingportion. The host cell is grown under conditions suitable for chimericprotein expression and assembly of the bacteriophage particles, and theassociation of the chimeric protein with the specific nucleotidesequence in the expression vector. In this embodiment, since the vectoris a bacteriophage, which replicates to produce a single-stranded DNA,the nucleotide binding portion preferably has an affinity forsingle-stranded DNA. Incorporation of the vector single-strandedDNA-chimeric protein complex into bacteriophage particles results in theassembly of the peptide display carrier package (PDCP), and display ofthe target peptide on the outer surface of the PDCP.

In this embodiment both of the required elements for producing peptidedisplay carrier packages are contained on the same vector. Incorporationof the DNA-chimeric protein complex into a peptide display carrierpackage (PDCP) is preferred as DNA degradation is prevented, largenumbers of PDCPs are produced per host cell, and the PDCPs are easilyseparated from the host cell without recourse to cell lysis.

In a more preferred embodiment, the vector of the is a phagemid vector(for example pUC119) where expression of the chimeric protein iscontrolled by an inducible promoter. In this embodiment the PDCP canonly be assembled following infection of the host cell with bothphagemid vector and helper phage. The transfected host cell is thencultivated under conditions suitable for chimeric protein expression andassembly of the bacteriophage particles.

In this embodiment the elements of the PDCP are provided by two separatevectors. The phagemid derived PDCP is superior to phagemid deriveddisplay packages disclosed in WO-A-92/01047 where a proportion ofpackages displaying bacteriophage coat protein fusion proteins willcontain the helper phage DNA, not the fusion protein DNA sequence. Inthe current invention, a PDCP can display the chimeric fusion proteinonly when the package contains the specific nucleotide motif recognisedby the nucleotide binding portion. In most embodiments this sequencewill be present on the same DNA segment that encodes the fusion protein.In addition, the prior art acknowledges that when mutant and wild typeproteins are co-expressed in the same bacterial cell, the wild typeprotein is produced preferentially. Thus, when the wild type helperphage, phage display system of WO-A-92/01047 is used, both wild typegene pIII and target peptide-gene pIII chimeric proteins are produced inthe same cell. The result of this is that the wild type gene pIIIprotein is preferentially packaged into bacteriophage particles, overthe chimeric protein. In the current invention, there is no competitionwith wild type bacteriophage coat proteins for packaging.

Desirably the target peptide is displayed in a location exposed to theexternal environment of the PDCP, after the PDCP particle has beenreleased from the host cell without recourse to cell lysis. The targetpeptide is then accessible for binding to its ligand. Thus, the targetpeptide may be located at or near the N-terminus or the C-terminus of anucleotide binding domain, for example the DNA binding domain of theoestrogen receptor.

The present invention also provides a method for screening a DNA libraryexpressing one or more polypeptide chains that are processed, folded andassembled in the periplasmic space to achieve biological activity. ThePDCP may be assembled by the following steps:

-   (a) Construction of N- or C-terminal DBD chimeric protein fusions in    a phagemid vector.    -   (i) When the target peptide is located at the N-terminus of the        nucleotide binding portion, a library of DNA sequences each        encoding a potential target peptide is cloned into an        appropriate location of an expression vector (i.e. behind an        appropriate promoter and translation sequences and a sequence        encoding a signal peptide leader directing transport of the        downstream fusion protein to the periplasmic space) and upstream        of the sequence encoding the nucleotide binding portion. In a        preferred embodiment the DNA sequence(s) of interest may be        joined, by a region of DNA encoding a flexible amino acid        linker, to the 5′-end of an oestrogen receptor DBD.    -   (ii) Alternatively, when the target peptide is located at the        C-terminus of the nucleotide binding domain, a library of DNA        sequences each encoding a potential target peptide is cloned        into the expression vector so that the nucleotide sequence        coding for the nucleotide binding portion is upstream of the        cloned DNA target peptide encoding sequences, said nucleotide        binding portion being positioned behind an appropriate promoter        and translation sequences and a sequence encoding a signal        peptide leader directing transport of the downstream fusion        protein to the periplasmic space. In a preferred embodiment, DNA        sequence(s) of interest may be joined, by a region of DNA        encoding a flexible amino acid linker oestrogen receptor DBD DNA        sequence.

Located on the expression vector is the specific HRE nucleotide sequencerecognised, and bound, by the oestrogen receptor DBD. In order to varythe number of chimeric proteins displayed on each PDCP particle, thissequence can be present as one or more copies in the vector.

-   (b) Incorporation into the PDCP. Non-lytic helper bacteriophage    infects host cells containing the expression vector. Preferred types    of bacteriophage include the filamentous phage fd, fl and M13. In a    more preferred embodiment the bacteriophage may be M13K07.

The protein(s) of interest are expressed and transported to theperiplasmic space, and the properly assembled proteins are incorporatedinto the PDCP particle by virtue of the high affinity interaction of theDBD with the specific target nucleotide sequence present on the phagemidvector DNA which is naturally packaged into phage particles in asingle-stranded form. The high affinity interaction between the DBDprotein and its specific target nucleotide sequence preventsdisplacement by bacteriophage coat proteins resulting in theincorporation of the protein(s) of interest onto the surface of the PDCPas it is extruded from the cell.

-   (c) Selection of the peptide of interest. Particles which display    the peptide of interest are then selected from the culture by    affinity enrichment techniques.

This is accomplished by means of a ligand specific for the protein ofinterest, such as an antigen if the protein of interest is an antibody.The ligand may be presented on a solid surface such as the surface of anELISA plate, or in solution. Repeating the affinity selection procedureprovides an enrichment of clones encoding the desired sequences, whichmay then be isolated for sequencing, further cloning and/or expression.

Numerous types of libraries of peptides fused to the DBD can be screenedunder this embodiment including:

-   -   (i) Random peptide sequences encoded by synthetic DNA of        variable length.    -   (ii) Single-chain Fv antibody fragments. These consist of the        antibody heavy and light chain variable region domains joined by        a flexible linker peptide to create a single-chain antigen        binding molecule.    -   (iii) Random fragments of naturally occurring proteins isolated        from a cell population containing an activity of interest.

In another embodiment the invention concerns methods for screening a DNAlibrary whose members require more than one chain for activity, asrequired by, for example, antibody Fab fragments for ligand binding. Inthis embodiment heavy or light chain antibody DNA is joined to anucleotide sequence encoding a DNA binding domain of, for example, theoestrogen receptor in a phagemid vector. Typically the antibody DNAlibrary sequences for either the heavy (VH and CH1) or light chain (VLand CL) genes are inserted in the 5′ region of the oestrogen receptorDBD DNA, behind an appropriate promoter and translation sequences and asequence encoding a signal peptide leader directing transport of thedownstream fusion protein to the periplasmic space.

Thus, a DBD fused to a DNA library member-encoded protein is producedand assembled in to the viral particle after infection withbacteriophage. The second and any subsequent chain(s) are expressedseparately either:

-   (a) from the same phagemid vector containing the DBD and the first    polypeptide fusion protein, or-   (b) from a separate region of DNA which may be present in the host    cell nucleus, or on a plasmid, phagemid or bacteriophage expression    vector that can co-exist, in the same host cell, with the first    expression vector, so as to be transported to the periplasm where    they assemble with the first chain that is fused to the DBD protein    as it exits the cell. Peptide display carrier packages (PDCP) which    encode the protein of interest can then be selected by means of a    ligand specific for the protein.

In yet another embodiment, the invention concerns screening libraries ofbi-functional peptide display carrier packages where two or moreactivities of interest are displayed on each PDCP. In this embodiment, afirst DNA library sequence(s) is inserted next to a first DNA bindingdomain (DBD) DNA sequence, for example the oestrogen receptor DBD, in anappropriate vector, behind an appropriate promoter and translationsequences and a sequence encoding a signal peptide leader directingtransport of this first chimeric protein to the periplasmic space. Asecond chimeric protein is also produced from the same, or separate,vector by inserting a second DNA library sequence(s) next to a secondDBD DNA sequence which is different from the first DBD DNA sequence, forexample the progesterone receptor DBD, behind an appropriate promoterand translation sequences and a sequence encoding a signal peptideleader. The first, or only, vector contains the specific HRE nucleotidesequences for both oestrogen and progesterone receptors. Expression ofthe two chimeric proteins, results in a PDCP with two different chimericproteins displayed. As an example, one chimeric protein could possess abinding activity for a particular ligand of interest, while the secondchimeric protein could possess an enzymatic activity. Binding by thePDCP to the ligand of the first chimeric protein could then be detectedby subsequent incubation with an appropriate substrate for the secondchimeric protein. In an alternative embodiment a bi-functional PDCP maybe created using a single DBD, by cloning one peptide at the 5′-end ofthe DBD, and a second peptide at the 3′-end of the DBD. Expression ofthis single bi-functional chimeric protein results in a PDCP with twodifferent activities.

We have investigated the possibility of screening libraries of peptides,fused to a DNA binding domain and displayed on the surface of a displaypackage, for particular peptides with a biological activity of interestand recovering the DNA encoding that activity. Surprisingly, bymanipulating the oestrogen receptor DNA binding domain in conjunctionwith M13 bacteriophage we have been able to construct novel particleswhich display large biologically functional molecules, that allowsenrichment of particles with the desired specificity.

The invention described herein provides a significant breakthrough inDNA library screening technology.

The invention will now be further described by reference to thenon-limiting examples and figures below.

DESCRIPTION OF FIGURES

FIG. 1 shows the pDM12 N-terminal fusion oestrogen receptor DNA bindingdomain expression vector nucleotide sequence (SEQ ID No 1), between theHindIII and EcoRI restriction sites, comprising a pelB leader secretionsequence (in italics) (SEQ ID No 2), multiple cloning site containingSfiI and NotI sites, flexible (glycine)₄-serine linker sequence (boxed),a fragment of the oestrogen receptor gene comprising amino acids 176-282(SEQ ID No 3) of the full length molecule, and the 38 base pairconsensus oestrogen receptor DNA binding domain HRE sequence.

FIG. 2 shows the OD_(450nm) ELISA data for negative control M13K07phage, and single-clone PDCP display culture supernatants (#1-4, seeExample 3) isolated by selection of the lymphocyte cDNA-pDM12 libraryagainst anti-human immunoglobulin kappa antibody.

FIG. 3 shows partial DNA (SEQ ID No 4) and amino acid (SEQ ID No 5)sequence for the human immunoglobulin kappa constant region (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest. 4^(th)edition. U.S. Department of Health and Human Services. 1987), and ELISApositive clones #2 (SEQ ID Nos 6 and 7) and #3 (SEQ ID Nos 8 and 9) fromFIG. 2 which confirms the presence of human kappa constant region DNAin-frame with the pelB leader sequence (pelB leader sequence isunderlined, the leader sequence cleavage site is indicated by an arrow).The differences in the 5′-end sequence demonstrates that these twoclones were selected independently from the library stock. The PCRprimer sequence is indicated in bold, clone #2 was originally amplifiedwith CDNAPCRBAK1 and clone #3 was amplified with CDNAPCRBAK2.

FIG. 4 shows the pDM14 N-terminal fusion oestrogen receptor DNA bindingdomain expression vector nucleotide sequence (SEQ ID No 10), between theHindIII and EcoRI restriction sites, comprising a pelB leader secretionsequence (in italics) (SEQ ID No 11), multiple cloning site containingSfiI and NotI sites, flexible (glycine)₄-serine linker sequence (boxed),a fragment of the oestrogen receptor gene comprising amino acids 176-282(see SEQ ID No 12) of the full length molecule, and the two 38 base pairoestrogen receptor DNA binding domain HRE sequences (HRE 1 and HRE 2).

FIG. 5 shows the pDM16 C-terminal fusion oestrogen receptor DNA bindingdomain expression vector nucleotide sequence (SEQ ID No 13), between theHindIII and EcoRI restriction sites, comprising a pelB leader secretionsequence (in italics), a fragment of the oestrogen receptor genecomprising amino acids 176-282 (SEQ ID No 14) of the full lengthmolecule, flexible (glycine)₄-serine linker sequence (boxed), multiplecloning site containing SfiI and NotI sites and the 38 base pairoestrogen receptor DNA binding domain HRE sequence.

FIG. 6 shows the OD_(450nm) ELISA data for N-cadherin-pDM16 C-terminaldisplay PDCP binding to anti-pan-cadherin monoclonal antibody in serialdilution ELISA as ampicillin resistance units (a.r.u.). Backgroundbinding of negative control M13K07 helper phage is also shown.

FIG. 7 shows the OD_(450nm) ELISA data for in vivo biotinylatedPCC-pDM16 C-terminal display PDCP binding to streptavidin in serialdilution ELISA as ampicillin resistance units (a.r.u.). Backgroundbinding of negative control M13K07 helper phage is also shown.

FIG. 8 shows the OD₄₅₀ nm ELISA data for a human scFv PDCP isolated froma human scFv PDCP display library selected against substance P. The PDCPwas tested against streptavidin (1), streptavidin-biotinylated substanceP (2), and streptavidin-biotinylated CGRP (3), in the presence (B) orabsence (A) of free substance P.

FIG. 9 shows the DNA (SEQ ID Nos 15 and 17) and amino acid (SEQ ID No 16and 18) sequence of the substance P binding scFv isolated from a humanscFv PDCP display library selected against substance P. Heavy chain (SEQID Nos 15 and 16) and light chain (SEQ ID Nos 17 and 18) variable regionsequence is shown with the CDRs underlined and highlighted in bold.

MATERIALS AND METHODS

The following procedures used by the present applicant are described inSambrook, J., et al., 1989 supra.: restriction enzyme digestion,ligation, preparation of electrocompetent cells, electroporation,analysis of restriction enzyme digestion products on agarose gels, DNApurification using phenol/chloroform, preparation of 2×TY medium andplates, preparation of ampicillin, kanamycin, IPTG (Isopropylβ-D-Thiogalactopyranoside) stock solutions, and preparation of phosphatebuffered saline.

Restriction enzymes, T4 DNA ligase and cDNA synthesis reagents(Superscript plasmid cDNA synthesis kit) were purchased from LifeTechnologies Ltd (Paisley, Scotland, U.K.). Oligonucleotides wereobtained from Cruachem Ltd (Glasgow, Scotland, U.K.), or GenosysBiotechnologies Ltd (Cambridge, U.K.). Taq DNA polymerase, Wizard SVplasmid DNA isolation kits, streptavidin coated magnetic beads and mRNAisolation reagents (PolyATract 1000) were obtained from Promega Ltd(Southampton, Hampshire, U.K.). Taqplus DNA polymerase was obtained fromStratagene Ltd (Cambridge, U.K.). PBS, BSA, streptavidin, substance Pand anti-pan cadherin antibody were obtained from SIGMA Ltd (Poole,Dorset, U.K.). Anti-M13-HRP conjugated antibody, Kanamycin resistantM13K07 helper bacteriophage and RNAguard were obtained from PharmaciaLtd (St. Albans, Herts, U.K.) and anti-human IgK antibody fromHarlan-Seralab (Loughborough, Leicestershire, U.K.) Biotinylatedsubstance P and biotinylated calcitonin gene related peptide (CGRP) wereobtained from Peninsula Laboratories (St. Helens, Merseyside, U.K.).

Specific embodiments of the invention are given below in Examples 1 to9.

EXAMPLE 1 Construction of a N-Terminal PDCP Display Phagemid VectorpDM12

The pDM12 vector was constructed by inserting an oestrogen receptor DNAbinding domain, modified by appropriate PCR primers, into a phagemidvector pDM6. The pDM6 vector is based on the pUC119 derived phagedisplay vector pHEN1 (Hoogenboom et al., 1991, Nucleic Acids Res. 19:4133-4137). It contains (Gly)₄Ser linker, Factor Xa cleavage site, afull length gene III, and streptavidin tag peptide sequence (Schmidt, T.G. and Skerra, A., 1993, Protein Engineering 6: 109-122), all of whichcan be removed by NotI-EcoRI digestion and agarose gel electrophoresis,leaving a pelB leader sequence, SfiI, NcoI and PstI restriction sitesupstream of the digested NotI site. The cloned DNA binding domain isunder the control of the lac promoter found in pUC119.

Preparation of pDM6

The pDM12 vector was constructed by inserting an oestrogen receptor DNAbinding domain, modified by appropriate PCR primers, into a phagemidvector pDM6. The pDM6 vector is based on the gene pIII phage displayvector pHEN1 (Hoogenboom et al., 1991, Nucleic Acids Res. 19:4133-4137), itself derived from pUC119 (Viera, J. and Messing, J., 1987,Methods in Enzymol. 153: 3-11). It was constructed by amplifying thepIII gene in pHEN1 with two oligonucleotides:

(SEQ ID No 19) PDM6BAK:5 -TTT TCT GCA GTA ATA GGC GGC CGC AGG GGG AGG AGGGTC CAT CGA AGG TCG CGA AGC AGA GAC TGT TGA AAG T- 3 and (SEQ ID No 20)PDM6FOR: 5 -TTT TGA ATT CTT ATT AAC CAC CGA ACT GCG GGT GACGCC AAG CGC TTG CGG CCG TTA AGA CTC CTT ATT ACG CAG-3.and cloning the PstI-EcoRI digested PCR product back into similarlydigested pHEN1, thereby removing the c-myc tag sequence and supE TAGcodon from pHEN1. The pDM6 vector contains a (Gly)₄Ser linker, Factor Xacleavage site, a full length gene III, and streptavidin tag peptidesequence (Schmidt, T. G. and Skerra, A., 1993, Protein Engineering 6:109-122), all of which can be removed by NotI-EcoRI digestion andagarose gel electrophoresis, leaving a pelB leader sequence, SfiI, NcoIand PstI restriction sites upstream of the digested NotI site. Thecloned DNA binding domain is under the control of the lac promoter foundin pUC119.

The oestrogen receptor DNA binding domain was isolated from cDNAprepared from human bone marrow (Clontech, Palo Alto, Calif., U.S.A.).cDNA can be prepared by many procedures well known to those skilled inthe art. As an example, the following method using a Superscript plasmidcDNA synthesis kit can be used:

(a) First Strand Synthesis.

5 μg of bone marrow mRNA, in 5 μl DEPC-treated water was thawed on iceand 2 μl (50 pmol) of cDNA synthesis primer(5′-AAAAGCGGCCGCACTGGCCTGAGAGA(N)₆-3′) (SEQ ID No 21) was added to themRNA and the mixture heated to 70° C. for 10 minutes, then snap-chilledon ice and spun briefly to collect the contents to the bottom of thetube. The following were then added to the tube:

1000 u/ml RNAguard 1 μl 5x first strand buffer 4 μl 0.1M DTT 2 μl 10 mMdNTPs 1 μl 200 u/μl Superscript II reverse transcriptase 5 μl

The mixture was mixed by pipetting gently and incubated at 37° C. for 1hour, then placed on ice.

(b) Second Strand Synthesis.

The following reagents were added to the first strand reaction:

DEPC-treated water 93 μl  5x second strand buffer 30 μl  10 mM dNTPs 3μl 10 u/μl E. coli DNA ligase 1 μl 10 u/μl E. coli DNA polymerase 4 μl 2 u/μl E. coli RNase H 1 μl

The reaction was vortex mixed and incubated at 16° C. for 2 hours. 2 μl(10u) of T4 DNA polymerase was added and incubation continued at 16° C.for 5 minutes. The reaction was placed on ice and 10 μl 0.5M EDTA added,then phenol-chloroform extracted, precipitated and vacuum dried.

(c) Sal I Adaptor Ligation.

The cDNA pellet was resuspended in 25 μl DEPC-treated water, andligation set up as follows.

cDNA 25 μl 5x T4 DNA ligase buffer 10 μl 1 μg/μl Sal I adapters* 10 μl1 u/μlT4 DNA ligase  5 μl *Sal I adapters: TCGACCCACGCGTCCG-3′(SEQ ID No 22) GGGTGCCGAGGC-5′ (SEQ ID No 23)

The ligation was mixed gently and incubated for 16 hours at 16° C., thenphenol-chloroform extracted, precipitated and vacuum dried. ThecDNA/adaptor pellet was resuspended in 41 μl of DEPC-treated water anddigested with 60 units of NotI at 37° C. for 2 hours, thenphenol-chloroform extracted, precipitated and vacuum dried. The cDNApellet was re-dissolved in 100 μl TEN buffer (10 mM Tris pH 7.5, 0.1 mMEDTA, 25 mM NaCl) and size fractionated using a Sephacryl S-500 HRcolumn to remove unligated adapters and small cDNA fragments (<400 bp)according to the manufacturers instructions. Fractions were checked byagarose gel electrophoresis and fractions containing cDNA less than 400base pairs discarded, while the remaining fractions were pooled.

(d) PCR Amplification of Oestrogen Receptor DNA Binding Domain.

The oestrogen receptor was PCR amplified from 5 μl (150-250 ng) of bonemarrow cDNA using 25 pmol of each of the primers pDM12FOR (SEQ ID No 24)(5′-AAAAGAATTCTGAATGTGTTATTTTAGCTCAGGTCACTCTGACCTGATTATCAAGACCCCACTTCACCCCCT)and pDM12BAK (SEQ ID No 25)(5′-AAAAGCGGCCGCAGGGGGAGGAGGGTCCATGGAATCTGCCAAGGAG-3′) in two 50 μlreactions containing 0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and1×PCR reaction buffer (10 mM Tris-HCl pH 9.0, 5 mM KCl, 0.01% TritonX®-100, 1.5 mM MgCl₂) (Promega Ltd, Southampton, U.K.). The pDM12FORprimer anneals to the 3′-end of the DNA binding domain of the oestrogenreceptor and incorporates two stop codons, the 38 base pair consensusoestrogen receptor HRE sequence, and an EcoRI restriction site. ThepDM12BAK primer anneals to the 5′-end of the DNA binding domain of theoestrogen receptor and incorporates the (Gly)₄Ser linker and the NotIrestriction site.

Reactions were overlaid with mineral oil and PCR carried out on a TechnePHC-3 thermal cycler for 30 cycles of 94° C., 1 minute; 65° C., 1minute; 72° C., 1 minute. Reaction products were electrophoresed on anagarose gel, excised and products purified from the gel using aGeneclean II kit according to the manufacturers instructions (Bio101, LaJolla, Calif., U.S.A.).

(e) Restriction Digestion and Ligation.

The PCR reaction appended NotI and EcoRI restriction sites, the(Gly)₄Ser linker, stop codons and the 38 base pair oestrogen receptortarget HRE nucleotide sequence to the oestrogen receptor DNA bindingdomain sequence (see FIG. 1). The DNA PCR fragment and the target pDM6vector (approximately 500 ng) were NotI and EcoRI digested for 1 hour at37° C., and DNA purified by agarose gel electrophoresis and extractionwith Geneclean II kit (Bio101, La Jolla, Calif., U.S.A.). The oestrogenreceptor DNA binding domain cassette was ligated into the NotI-EcoRIdigested pDM6 vector overnight at 16° C., phenol/chloroform extractedand precipitated then electroporated into TG1 E. coli (genotype: K12,(Δlac-pro), supE, thi, hsD5/F′traD36, proA⁺B⁺, LacI^(q), LacZΔ15) andplated onto 2×TY agar plates supplemented with 1% glucose and 100 μg/mlampicillin. Colonies were allowed to grow overnight at 37° C. Individualcolonies were picked into 5 ml 2×TY supplemented with 1% glucose and 100μg/ml ampicillin and grown overnight at 37° C. Double stranded phagemidDNA was isolated with a Wizard SV plasmid DNA isolation kit and thesequence confirmed with a Prism dyedeoxy cycle sequencing kit(Perkin-Elmer, Warrington, Lancashire, U.K.) using M13FOR (SEQ ID No 26)(5′-GTAAAACGACGGCCAGT) and M13REV (SEQ ID No 27)(5′-GGATAACAATTTCACACAGG) oligonucleotides. The pDM12 PDCP displayvector DNA sequence between the HindIII and EcoRI restriction sites isshown in FIG. 1.

EXAMPLE 2 Insertion of a Random-Primed Human Lymphocyte cDNA into pDM12and Preparation of a Master PDCP Stock

Libraries of peptides can be constructed by many methods known to thoseskilled in the art. The example given describes a method forconstructing a peptide library from randomly primed cDNA, prepared frommRNA isolated from a partially purified cell population.

mRNA was isolated from approximately 10⁹ human peripheral bloodlymphocytes using a polyATract 1000 mRNA isolation kit (Promega,Southampton, UK). The cell pellet was resuspended in 4 ml extractionbuffer (4M guanidine thiocyanate, 25 mM sodium citrate pH 7.1, 2%β-mercaptoethanol). 8 ml of pre-heated (70° C.) dilution buffer (6×SSC,10 mM Tris pH 7.4, 1 mM EDTA, 0.25% SDS, 1% β-mercaptoethanol) was addedto the homogenate and mixed thoroughly by inversion. 10 μl ofbiotinylated oligo-dT (50 pmol/μl) was added, mixed and the mixtureincubated at 70° C. for 5 minutes. The lymphocyte cell lysate wastransferred to 6×2 ml sterile tubes and spun at 13,000 rpm in amicrocentrifuge for ten minutes at ambient temperature to produce acleared lysate. During this centrifugation, streptavidin coated magneticbeads were resuspended and 6 ml transferred to a sterile 50 ml Falcontube, then placed in the magnetic stand in a horizontal position untilall the beads were captured. The supernatant was carefully poured offand beads resuspended in 6 ml 0.5×SSC, then the capture repeated. Thiswash was repeated 3 times, and beads resuspended in a final volume of 6ml 0.5×SSC. The cleared lysate was added to the washed beads, mixed byinversion and incubated at ambient temperature for 2 minutes, then beadscaptured in the magnetic stand in a horizontal position. The beads wereresuspended gently in 2 ml 0.5×SSC and transferred to a sterile 2 mlscrewtop tube, then captured again in the vertical position, and thewash solution discarded. This wash was repeated twice more. 1 ml ofDEPC-treated water was added to the beads and mixed gently. The beadswere again captured and the eluted mRNA transferred to a sterile tube.501 was electrophoresed to check the quality and quantity of mRNA, whilethe remainder was precipitated with 0.1 volumes 3M sodium acetate andthree volumes absolute ethanol at −80° C. overnight in 4 aliquots insterile 1.5 ml screwtop tubes.

Double stranded cDNA was synthesised as described in Example 1 using 5μg of lymphocyte mRNA as template. The cDNA was PCR amplified usingoligonucleotides CDNAPCRFOR (SEQ ID No 28)(5′-AAAGCGGCCGCACTGGCCTGAGAGA), which anneals to the cDNA synthesisoligonucleotide described in Example 1 which is present at the 3′-end ofall synthesised cDNA molecules incorporates a NotI restriction site, andan equimolar mixture of CDNAPCRBAK1, CDNAPCRBAK2 and CDNAPCRBAK3.CDNAPCRBAK1: (SEQ ID No 29)5′-AAAAGGCCCAGCCGGCCATGGCCCAGCCCACCACGCGTCCG, CDNAPCRBAK2: (SEQ ID No30) 5′-AAAAGGCCCAGCCGGCCATGGCCCAGTCCCACCACGCGTCCG, CDNAPCRBAK3: (SEQ IDNo 31) 5′-AAAAGGCCCAGCCGGCCATGGCCCAGTACCCACCACGCGTCCG), all three ofwhich anneal to the SalI adaptor sequence found at the 5′-end of thecDNA and incorporate a SfiI restriction site at the cDNA 5′-end. Ten PCRreactions were carried out using 2 μl of cDNA (50 ng) per reaction asdescribed in Example 1 using 25 cycles of 94° C., 1 minute; 60° C., 1minute; 72° C., 2 minutes. The reactions were pooled and a 20 μl aliquotchecked by agarose gel electrophoresis, the remainder wasphenol/chloroform extracted and ethanol precipitated and resuspended in100 μl sterile water. 5 μg of pDM12 vector DNA and lymphocyte cDNA PCRproduct were SfiI-NotI digested phenol/chloroform extracted and smallDNA fragments removed by size selection on Chromaspin 1000 spin columns(Clontech, Palo Alto, Calif., U.S.A.) by centrifugation at 700 g for 2minutes at room temperature. Digested pDM12 and lymphocyte cDNA wereethanol precipitated and ligated together for 16 hours at 16° C. Theligated DNA was precipitated and electroporated in to TG1 E. coli. Cellswere grown in 1 ml SOC medium per cuvette used for 1 hour at 37° C., andplated onto 2×TY agar plates supplemented with 1% glucose and 100 μg/mlampicillin. 10⁻⁴, 10⁻⁵ and 10⁻⁶ dilutions of the electroporated bacteriawere also plated to assess library size. Colonies were allowed to growovernight at 30° C. 2×10⁸ ampicillin resistant colonies were recoveredon the agar plates.

The bacteria were then scraped off the plates into 40 ml 2×TY brothsupplemented with 20% glycerol, 1% glucose and 100 μg/ml ampicillin. 5ml was added to a 20 ml 2×TY culture broth supplemented with 1% glucoseand 100 μg/ml ampicillin and infected with 10¹¹ kanamycin resistanceunits (kru) M13K07 helper phage at 37° C. for 30 minutes withoutshaking, then for 30 minutes with shaking at 200 rpm. Infected bacteriawere transferred to 200 ml 2×TY broth supplemented with 25 μg/mlkanamycin, 100 μg/ml ampicillin, and 20 μM IPTG, then incubatedovernight at 37° C., shaking at 200 rpm. Bacteria were pelleted at 4000rpm for 20 minutes in 50 ml Falcon tubes, and 40 ml 2.5M NaCl/20% PEG6000 was added to 200 ml of particle supernatant, mixed vigorously andincubated on ice for 1 hour to precipitate PDCP particles. Particleswere pelleted at 11000 rpm for 30 minutes in 250 ml Oakridge tubes at 4°C. in a Sorvall RC5B centrifuge, then resuspended in 2 ml PBS bufferafter removing all traces of PEG/NaCl with a pipette, then bacterialdebris removed by a 5 minute 13500 rpm spin in a microcentrifuge. Thesupernatent was filtered through a 0.45 μm polysulfone syringe filterand stored at −20° C.

EXAMPLE 3 Isolation of Human Immunoglobulin Kappa Light Chains byRepeated Rounds of Selection Against Anti-Human Kappa Antibody

For the first round of library selection a 70×11 mm NUNC MaxisorpImmunotube (Life Technologies, Paisley, Scotland U.K.) was coated with2.5 ml of 10 μg/ml of anti-human kappa antibody (Seralab, Crawley Down,Sussex, U.K.) in PBS for 2 hours at 37° C. The tube was rinsed threetimes with PBS (fill & empty) and blocked with 3 ml PBS/2% BSA for 2hours at 37° C. and washed as before. 4×10¹² a.r.u. of pDM12-lymphocytecDNA PDCP stock was added in 2 ml 2% BSA/PBS/0.05% Tween 20, andincubated for 30 minutes on a blood mixer, then for 90 minutes standingat ambient temperature. The tube was washed ten times with PBS/0.1%Tween 20, then a further ten times with PBS only. Bound particles wereeluted in 1 ml of freshly prepared 0.1M triethylamine for 10 minutes atambient temperature on a blood mixer. Eluted particles were transferredto 0.5 ml 1M Tris pH 7.4, vortex mixed briefly and transferred to ice.

Neutralised particles were added to 10 ml log phase TG1 E coli bacteria(optical density: OD₆₀₀ nm 0.3-0.5) and incubated at 37° C. withoutshaking for 30 minutes, then with shaking at 200 rpm for 30 minutes.10⁻³, 10⁻⁴ & 10⁻⁵ dilutions of the infected culture were prepared toestimate the number of particles recovered, and the remainder was spunat 4000 rpm for 10 minutes, and the pellet resuspended in 300 μl 2×TYmedium by vortex mixing. Bacteria were plated onto 2×TY agar platessupplemented with 1% glucose and 100 μg/ml ampicillin. Colonies wereallowed to grow overnight at 30° C.

A PDCP stock was prepared from the bacteria recovered from the firstround of selection, as described in Example 2 from a 100 ml overnightculture. 250 μl of the round 1 amplified PDCP stock was then selectedagainst anti-human kappa antibody as described above with the tube waswashed twelve times with PBS/0.1% Tween 20, then a further twelve timeswith PBS only.

To identify selected clones, eighty-eight individual clones recoveredfrom the second round of selection were then tested by ELISA for bindingto anti-human kappa antibody. Individual colonies were picked into 100μl 2×TY supplemented with 100 μg/ml ampicillin and 1% glucose in 96-wellplates (Costar) and incubated at 37° C. and shaken at 200 rpm for 4hours. 25 μl of each culture was transferred to a fresh 96-well plate,containing 25 μl/well of the same medium plus 10⁷ k.r.u. M13K07kanamycin resistant helper phage and incubated at 37° C. for 30 minuteswithout shaking, then incubated at 37° C. and shaken at 200 rpm for afurther 30 minutes. 160 μl of 2×TY supplemented with 100 μg/mlampicillin, 25 μg/ml kanamycin, and 20 μM IPTG was added to each welland particle amplification continued for 16 hours at 37° C. whileshaking at 200 rpm. Bacterial cultures were spun in microtitre platecarriers at 2000 g for 10 minutes at 4° C. in a benchtop centrifuge topellet bacteria and culture supernatant used for ELISA.

A Dynatech Immulon 4 ELISA plate was coated with 200 ng/well anti-humankappa antibody in 100 μl/well PBS for one hour at 37° C. The plate waswashed 2×200 μl/well PBS and blocked for 1 hour at 37° C. with 200μl/well 2% BSA/PBS and then washed 2×200 μl/well PBS. 50 μl PDCP culturesupernatant was added to each well containing 50 μl/well 4% BSA/PBS/0.1%Tween 20, and allowed to bind for 1 hour at ambient temperature. Theplate was washed three times with 200 μl/well PBS/0.1% Tween 20, thenthree times with 200 μl/well PBS. Bound PDCPs were detected with 100μl/well, 1:5000 diluted anti-M13-HRP conjugate (Pharmacia) in 2%BSA/PBS/0.05% Tween 20 for 1 hour at ambient temperature and the platewashed six times as above. The plate was developed for 5 minutes atambient temperature with 100 μl/well freshly prepared TMB(3,3′,5,5′-Tetramethylbenzidine) substrate buffer (0.005% H₂O₂, 0.1mg/ml TMB in 24 mM citric acid/52 mM sodium phosphate buffer pH 5.2).The reaction was stopped with 100 μl/well 12.5% H₂SO₄ and read at 450nm. (ELISA data for binding clones is shown in FIG. 2). These cloneswere then sequenced with M13REV primer (SEQ ID No 27) as in Example 1.The sequence of two of the clones isolated is shown in FIG. 3 (see SEQID Nos 7 to 10).

EXAMPLE 4 Construction of the pDM14 N-Terminal Display Vector

It would be useful to design vectors that contain a second DBD bindingsequence, such as a second oestrogen receptor HRE sequence, thusallowing the display of increased numbers of peptides per PDCP. Peale etal. (1988, Proc. Natl. Acad. Sci. USA 85: 1038-1042) describe a numberof oestrogen receptor HRE sequences. These sequences were used to definean HRE sequence, which differs from that cloned in pDM12, which we usedto create a second N-terminal display vector (pDM14). Theoligonucleotide:5′-AAAAGAATTCGAGGTTACATTAACTTTGTTCCGGTCAGACTGACCCAAGTCGACCTGAATGTGTTATTTTAG-3,(SEQ ID No 32) was synthesised and used to mutagenise pDM12 by PCR withpDM12BAK oligonucleotide as described in Example 1 using 100 ng pDM12vector DNA as template. The resulting DNA fragment, which contained theoestrogen receptor DBD and two HRE sequences separated by a SalIrestriction enzyme site, was NotI-EcoRI restriction enzyme digested andcloned into NotI-EcoRI digested pDM12 vector DNA as described in Example1 to create pDM14. The sequence of pDM14 between the HindIII and EcoRIrestriction enzyme sites was checked by DNA sequencing. The final vectorsequence between these two sites is shown in FIG. 4 (see SEQ ID Nos 11and 12).

EXAMPLE 5 Construction of the pDM16 C-Terminal Display Vector

In order to demonstrate the display of peptides fused to the C-terminusof a DBD on a PDCP a suitable vector, pDM16, was created.

In pDM16 the pelB leader DNA sequence is fused directly to the oestrogenreceptor DBD sequence removing the multiple cloning sites and theGly₄Ser linker DNA sequence found in pDM12 and pDM14, which are appendedto the C-terminal end of the DBD sequence upstream of the HRE DNAsequence.

To create this vector two separate PCR reactions were carried out on aTechne Progene thermal cycler for 30 cycles of 94° C., 1 minute; 60° C.,1 minute; 72° C., 1 minute. Reaction products were electrophoresed on anagarose gel, excised and products purified from the gel using a Mermaidor Geneclean II kit, respectively, according to the manufacturersinstructions (Bio101, La Jolla, Calif., U.S.A.).

In the first, the 5′-untranslated region and pelB leader DNA sequencewas amplified from 100 ng of pDM12 vector DNA using 50 pmol of each ofthe oligonucleotides pelBFOR (SEQ ID No 33)(5′-CCTTGGCAGATTCCATCTCGGCCATTGCCGGC-3′) and M13REV (SEQ ID NO 27) (seeabove) in a 100 μl reaction containing 0.1 mM dNTPs, 2.5 units TaqplusDNA polymerase, and 1× High Salt PCR reaction buffer (20 mM Tris-HCl pH9.2, 60 mM KCl, 2 mM MgCl₂) (Stratagene Ltd, Cambridge, U.K.).

In the second, the 3′-end of the pelB leader sequence and the oestrogenreceptor DBD was amplified from 100 ng of pDM12 vector DNA using 50 pmolof each of the oligonucleotides pelBBAK (SEQ ID No 34)(5′-CCGGCAATGGCCGAGATGGAATCTGCCAAGG-3′) and pDM16FOR (SEQ ID No 35)(5′-TTTTGTCGACTCAATCAGTTATGCGGCCGCCAGCTGCAGGAGGGCCGGCTGGGCCGACCCTCCTCCCCCAGACCCCACTTCACCCC-3′)in a 100 μl reaction containing 0.1 mM dNTPs, 2.5 units Taqplus DNApolymerase, and 1× High Salt PCR reaction buffer (Stratagene Ltd,Cambridge, U.K.). Following gel purification both products were mixedtogether and a final round of PCR amplification carried out to link thetwo products together as described above, in a 100 μl reactioncontaining 0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and 1×PCRreaction buffer (10 mM Tris-HCl pH 9.0, 5 mM KCl, 0.01% Triton X®-100,1.5 mM MgCl₂) (Promega Ltd, Southampton, U.K.).

The resulting DNA fragment, was HindIII-SalI restriction enzyme digestedand cloned into HindIII-SalI digested pDM14 vector DNA as described inExample 1 to create pDM16. The sequence of pDM16 between the HindIII andEcoRI restriction enzyme sites was checked by DNA sequencing. The finalvector sequence between these two sites is shown in FIG. 5 (see SEQ IDNos 13 and 14).

EXAMPLE 6 Display of the C-Terminal Fragment of Human N-Cadherin on theSurface of a PDCP

cDNA libraries of peptides can be constructed by many methods known tothose skilled in the art. One commonly used method for constructing apeptide library uses oligo dT primed cDNA, prepared from polyA+ mRNA. Inthis method the first-strand synthesis is carried out using anoligonucleotide which anneals to the 3′-end polyA tail of the mRNAcomposed of T_(n) (where n is normally between 10 and 20 bases) and arestriction enzyme site such as NotI to facilitate cloning of cDNA. ThecDNA cloned by this method is normally composed of the polyA tail, the3′-end untranslated region and the C-terminal coding region of theprotein. As an example of the C-terminal display of peptides on a PDCP,a human cDNA isolated from a library constructed by the above method waschosen.

The protein N-cadherin is a cell surface molecule involved in cell-celladhesion. The C-terminal cytoplasmic domain of the human protein(Genbank database accession number: M34064) is recognised by acommercially available monoclonal antibody which was raised against theC-terminal 23 amino acids of chicken N-cadherin (SIGMA catalogue number:C-1821). The 1.4 kb human cDNA fragment encoding the C-terminal 99 aminoacids, 3′-untranslated region and polyA tail (NotI site present at the3′-end of the polyA tail) was amplified from approximately 20 ngpDM7-NCAD#C with 25 pmol of each oligonucleotide M13FOR (SEQ ID No 26)and CDNPCRBAK1 (SEQ ID No 29) (see above) in a 501 reaction containing0.1 mM dNTPs, 2.5 units Taqplus DNA polymerase, and 1× High Salt PCRreaction buffer (20 mM Tris-HCl pH 9.2, 60 mM KCl, 2 mM MgCl₂)(Stratagene Ltd, Cambridge, U.K.) on a Techne Progene thermal cycler for30 cycles of 94° C., 1 minute; 60° C., 1 minute; 72° C., 1 minute.Following gel purification and digestion with SfiI and NotI restrictionenzymes, the PCR product was cloned into pDM16 using an analogousprotocol as described in Example 1.

Clones containing inserts were identified by ELISA of 96 individual PDCPcultures prepared as described in Example 3. A Dynatech Immulon 4 ELISAplate was coated with 1:250 diluted anti-pan cadherin monoclonalantibody in 100 μl/well PBS overnight at 4° C. The plate was washed3×200 μl/well PBS and blocked for 1 hour at 37° C. with 200 μl/well 2%Marvel non-fat milk powder/PBS and then washed 2×200 μl/well PBS. 50 μlPDCP culture supernatant was added to each well containing 50 μl/well 4%Marvel/PBS, and allowed to bind for 1 hour at ambient temperature. Theplate was washed three times with 200 μl/well PBS/0.1% Tween 20, thenthree times with 200 μl/well PBS. Bound PDCPs were detected with 100μl/well, 1:5000 diluted anti-M13-HRP conjugate (Pharmacia) in 2%Marvel/PBS for 1 hour at ambient temperature and the plate washed sixtimes as above. The plate was developed for 15 minutes at ambienttemperature with 100 μl/well freshly prepared TMB(3,3′,5,5′-Tetramethylbenzidine) substrate buffer (0.005% H₂O₂, 0.1mg/ml TMB in 24 mM citric acid/52 mM sodium phosphate buffer pH 5.2).The reaction was stopped with 100 μl/well 12.5% H₂SO₄ and read at 450nm. The nucleotide sequence of an ELISA positive clone insert and DBDjunction was checked by DNA sequencing using oligonucleotides M13FOR(SEQ ID No 26) (see Example 1) and ORSEQBAK (SEQ ID No 36)(5′-TGTTGAAACACAAGCGCCAG-3′).

A fifty-fold concentrated stock of C-terminal N-cadherin PDCP particleswas prepared by growing the un-infected TG1 clone in 1 ml 2×TY culturebroth supplemented with 1% glucose and 100 g/ml ampicillin for fivehours at 37° C., shaking at 200 rpm and infecting with 108 kanamycinresistance units (kru) M13K07 helper phage at 37° C. for 30 minuteswithout shaking, then for 30 minutes with shaking at 200 rpm. Infectedbacteria were transferred to 20 ml 2×TY broth supplemented with 25 μg/mlkanamycin, 100 g/ml ampicillin, and 20 μM IPTG, then incubated overnightat 30° C., shaking at 200 rpm. Bacteria were pelleted at 4000 rpm for 20minutes in 50 ml Falcon tubes, and 4 ml 2.5M NaCl/20% PEG 6000 was addedto 20 ml of PDCP supernatant, mixed vigorously and incubated on ice for1 hour to precipitate particles.

The particles were pelleted at 11000 rpm for 30 minutes in 50 mlOakridge tubes at 4° C. in a Sorvall RC5B centrifuge, then resuspendedin PBS buffer after removing all traces of PEG/NaCl with a pipette, thenbacterial debris removed by a 5 minute 13500 rpm spin in amicrocentrifuge. The supernatant was filtered through a 0.45 μmpolysulfone syringe filter. The concentrated stock was two-fold seriallydiluted and used in ELISA against plates coated with anti-pan-cadherinantibody as described above (see FIG. 6).

This example demonstrates the principle of C-terminal display usingPDCPs, that C-terminal DBD-peptide fusion PDCPs can be made which can bedetected in ELISA, and the possibility that oligo dT primed cDNAlibraries may be displayed using this method.

EXAMPLE 7 Display of In Vivo Biotinylated C-Terminal Domain of HumanPropionyl CoA Carboxylase on the Surface of a PDCP

Example 6 shows that the C-terminal domain of human N-cadherin can beexpressed on the surface of a PDCP as a C-terminal fusion with the DBD.Here it is shown that the C-terminal domain of another human proteinpropionyl CoA carboxylase alpha chain (Genbank accession number: X14608)can similarly be displayed, suggesting that this methodology may begeneral.

The alpha sub-unit of propionyl CoA carboxylase alpha chain (PCC)contains 703 amino acids and is normally biotinylated at position 669.It is demonstrated that the PCC peptide displayed on the PDCP isbiotinylated, as has been shown to occur when the protein is expressedin bacterial cells (Leon-Del-Rio & Gravel; 1994, J. Biol. Chem. 37,22964-22968).

The 0.8 kb human cDNA fragment of PCC alpha encoding the C-terminal 95amino acids, 3′-untranslated region and polyA tail (NotI site present atthe 3′-end of the polyA tail) was amplified and cloned into pDM16 fromapproximately 20 ng pDM7-PCC#C with 25 pmol of each oligonucleotideM13FOR (SEQ ID No 26) and CDNPCRBAK1 (SEQ ID No 29) as described inExample 6.

Clones containing inserts were identified by ELISA as described inExample 6, except that streptavidin was coated onto the ELISA plate at250 ng/well, in place of the anti-cadherin antibody. The nucleotidesequence of an ELISA positive clone insert and DBD junction was checkedby DNA sequencing using oligonucleotides M13FOR (SEQ ID No 26) andORSEQBAK (SEQ ID No 36) (see above). A fifty-fold concentrated stock ofC-terminal PCC PDCP particles was prepared and tested in ELISA againststreptavidin as described in Example 6 (see FIG. 7).

This example shows not only that the peptide can be displayed as aC-terminal fusion on a PDCP, but also that in vivo modified peptides canbe displayed.

EXAMPLE 8 Construction of a Human scFv PDCP Display Library

This example describes the generation of a human antibody library ofscFvs made from an un-immunised human. The overall strategy for the PCRassembly of scFv fragments is similar to that employed by Marks, J. D.et al. 1991, J. Mol. Biol. 222: 581-597. The antibody geneoligonucleotides used to construct the library are derived from theMarke et al., paper and from sequence data extracted from the Kabatdatabase (Kabat, E. A. et al., Sequences of Proteins of ImmunologicalInterest. 4^(th) edition. U.S. Department of Health and Human Services.1987). The three linker oligonucleotides are described by Zhou et al.(1994, Nucleic Acids Res., 22: 888-889), all oligonucleotides used aredetailed in Table 1.

First, mRNA was isolated from peripheral blood lymphocytes and cDNAprepared for four repertoires of antibody genes IgD, IgM, Igκ, and Igλ,using four separate cDNA synthesis primers. VH genes were amplified fromIgD and IgM primed cDNA, and VL genes were amplified from Igκ and Igλprimed cDNA. A portion of each set of amplified heavy chain or lightchain DNA was then spliced with a separate piece of linker DNA encodingthe 15 amino acids (Gly₄ Ser)₃ (Huston, J. S. et al. 1989, Gene, 77:61). The 3′-end of the VH PCR products and the 5′-end of the VL PCRproducts overlap the linker sequence as a result of incorporating linkersequence in the JH, Vκ and Vλ family primer sets (Table 1). EachVH-linker or linker-VL DNA product was then spliced with either VH or VLDNA to produce the primary scFv product in a VH-linker-VL configuration.This scFv product was then amplified and cloned into pDM12 as aSfiI-NotI fragment, electroporated into TG1 and a concentrated PDCPstock prepared.

mRNA Isolation and cDNA Synthesis.

Human lymphocyte mRNA was purified as described in Example 2. SeparatecDNA reactions were performed with IGDCDNAFOR (SEQ ID No 37), IGMCDNAFOR(SEQ ID No 38), IGKCDNAFOR (SEQ ID No 39) and IGλCDNAFOR (SEQ ID No 40)oligonucleotides. 50 pmol of each primer was added to approximately 5 μgof mRNA in 20 μl of nuclease free water and heated to 70° C. for 5minutes and cooled rapidly on ice, then made up to a final reactionvolume of 100 μl containing 50 mM Tris pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10mM DTT, 0.5 mM dNTPs, and 2000 units of Superscript II reversetranscriptase (Life Technologies, Paisley, Scotland, U.K.). Thereactions were incubated at 37° C. for two hours, then heated to 95° C.for 5 minutes.

Primary PCRs.

For the primary PCR amplifications separate amplifications were set upfor each family specific primer with either an equimolar mixture of theJHFOR primer set (SEQ ID Nos 41 to 44) for IgM and IgD cDNA, or withSCFVKFOR (SEQ ID No 51) or SCFVXFOR (SEQ ID No 52) for IgK or Igλ cDNArespectively e.g. VH1BAK and JHFOR set; Vκ2BAK (SEQ ID No 54) andSCFVκFOR (SEQ ID No 51); Vλ3aBAK (SEQ ID No 66) and SCFVλFOR (SEQ ID No52) etc. Thus, for IgM, IgD and Igκ cDNA six separate reactions were setup, and seven for Igλ cDNA. A 50 μl reaction mixture was preparedcontaining 2 μl cDNA, 25 pmol of the appropriate FOR and BAK primers,0.1 mM dNTPs, 2.5 units Taqplus DNA polymerase, and 1× High Salt PCRreaction buffer (20 mM Tris-HCl pH 9.2, 60 mM KCl, 2 mM MgCl₂)(Stratagene Ltd, Cambridge, U.K.). Reactions were amplified on a TechneProgene thermal cycler for 30 cycles of 94° C., 1 minute; 60° C., 1minute; 72° C., 2 minutes, followed by 10 minutes at 72° C. Fiftymicroliters of all 25 reaction products were electrophoresed on anagarose gel, excised and products purified from the gel using aGeneclean II kit according to the manufacturers instructions (Bio101, LaJolla, Calif., U.S.A.). All sets of IgD, IgM, IgK or Igλ reactionproducts were pooled to produce VH or VL DNA sets for each of the fourrepertoires. These were then adjusted to approximately 20 ng/μl.

Preparation of Linker.

Linker product was prepared from eight 100 μl reactions containing 5 ngLINKAMP3T (SEQ ID No 76) template oligonucleotide, 50 pmol of LINKAMP3(SEQ ID No 74) and LINKAMP5 (SEQ ID No 75) primers, 0.1 mM dNTPs, 2.5units Taqplus DNA polymerase, and 1× High Salt PCR reaction buffer (20mM Tris-HCl pH 9.2, 60 mM KCl, 2 mM MgCl₂) (Stratagene Ltd, Cambridge,U.K.). Reactions were amplified on a Techne Progene thermal cycler for30 cycles of 94° C., 1 minute; 60° C., 1 minute; 72° C., 1 minute,followed by 10 minutes at 72° C. All reaction product waselectrophoresed on a 2% low melting point agarose gel, excised andproducts purified from the gel using a Mermaid kit according to themanufacturers instructions (Bio101, La Jolla, Calif., U.S.A.) andadjusted to 5 ng/μl.

First Stage Linking.

Four linking reactions were prepared for each repertoire using 20 ng ofVH or VL DNA with 5 ng of Linker DNA in 100 μl reactions containing (forIgM or IgD VH) 50 pmol of LINKAMPFOR and VH1-6BAK set, or, 50 pmolLINKAMPBAK and either SCFVκFOR (Igκ) or SCFVλFOR (Igλ), 0.1 mM dNTPs,2.5 units Taq DNA polymerase, and 1×PCR reaction buffer (10 mM Tris-HClpH 9.0, 5 mM KCl, 0.01% Triton X®-100, 1.5 mM MgCl₂) (Promega Ltd,Southampton, U.K.). Reactions were amplified on a Techne Progene thermalcycler for 30 cycles of 94° C., 1 minute; 60° C., 1 minute; 72° C., 2minutes, followed by 10 minutes at 72° C. Reaction products wereelectrophoresed on an agarose gel, excised and products purified fromthe gel using a Geneclean II kit according to the manufacturersinstructions (Bio101, La Jolla, Calif., U.S.A.) and adjusted to 20ng/μl.

Final Linking and Reamplification.

To prepare the final scFv DNA products, five 100 μl reactions wereperformed for VH-LINKER plus VL DNA, and, five 100 μl reactions wereperformed for VH plus LINKER-VL DNA for each of the four finalrepertoires (IgM VH-VK, VH-Vλ; IgD VH-VK, VH-Vλ) as described in step(d) above using 20 ng of each component DNA as template. Reactionproducts were electrophoresed on an agarose gel, excised and productspurified from the gel using a Geneclean II kit according to themanufacturers instructions (Bio101, La Jolla, Calif., U.S.A.) andadjusted to 20 ng/μl. Each of the four repertoires was then re-amplifiedin a 100 μl reaction volume containing 2 ng of each linked product, with50 pmol VHBAK1-6 (SEQ ID Nos 53 to 58) and either the JKFOR (SEQ ID Nos66 to 70) or JλFOR (SEQ ID Nos 71 to 73) primer sets, in the presence of0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and 1×PCR reaction buffer(10 mM Tris-HCl pH 9.0, 5 mM KCl, 0.01% Triton XO-100, 1.5 mM MgCl₂)(Promega Ltd, Southampton, U.K.). Thirty reactions were performed perrepertoire to generate enough DNA for cloning. Reactions were amplifiedon a Techne Progene thermal cycler for 25 cycles of 94° C., 1 minute;65° C., 1 minute; 72° C., 2 minutes, followed by 10 minutes at 72° C.Reaction products were phenol-chloroform extracted, ethanolprecipitated, vacuum dried and re-suspended in 80 μl nuclease freewater.

Cloning into pDM12.

Each of the four repertoires was SfiI-NotI digested, and electrophoresedon an agarose gel, excised and products purified from the gel using aGeneclean II kit according to the manufacturers instructions (Bio101, LaJolla, Calif., U.S.A.). Each of the four repertoires was ligatedovernight at 16° C. in 140 μl with 10 μg of SfiI-NotI cut pDM12 preparedas in Example 2, and 12 units of T4 DNA ligase (Life Technologies,Paisley, Scotland, U.K.). After incubation the ligations were adjustedto 200 μl with nuclease free water, and DNA precipitated with 1 μl 20mg/ml glycogen, 100 μl 7.5M ammonium acetate and 900 μl ice-cold (−20°C.) absolute ethanol, vortex mixed and spun at 13,000 rpm for 20 minutesin a microfuge to pellet DNA. The pellets were washed with 500 μlice-cold 70% ethanol by centrifugation at 13,000 rpm for 2 minutes, thenvacuum dried and re-suspended in 10 μl DEPC-treated water. 1 μl aliquotsof each repertoire was electroporated into 80 μl E. coli (TG1). Cellswere grown in 1 ml SOC medium per cuvette used for 1 hour at 37° C., andplated onto 2×TY agar plates supplemented with 1% glucose and 100 μg/mlampicillin. 10⁻⁴, 10⁻⁵ and 10⁻⁶ dilutions of the electroporated bacteriawere also plated to assess library size. Colonies were allowed to growovernight at 30° C. Cloning into SfiI-NotI digested pDM12 yielded anIgM-κ/λ repertoire of 1.16×10⁹ clones, and an IgD-κ/λ repertoire of1.21×10⁹ clones.

Preparation of PDCP Stock.

Separate PDCP stocks were prepared for each repertoire library. Thebacteria were then scraped off the plates into 30 ml 2×TY brothsupplemented with 20% glycerol, 1% glucose and 100 μg/ml ampicillin. 3ml was added to a 50 ml 2×TY culture broth supplemented with 1% glucoseand 100 g/ml ampicillin and infected with 10¹¹ kanamycin resistanceunits (kru) M13K07 helper phage at 37° C. for 30 minutes withoutshaking, then for 30 minutes with shaking at 200 rpm. Infected bacteriawere transferred to 500 ml 2×TY broth supplemented with 25 μg/mlkanamycin, 100 μg/ml ampicillin, and 20 μM IPTG, then incubatedovernight at 30° C., shaking at 200 rpm. Bacteria were pelleted at 4000rpm for 20 minutes in 50 ml Falcon tubes, and 80 ml 2.5M NaCl/20% PEG6000 was added to 400 ml of particle supernatant, mixed vigorously andincubated on ice for 1 hour to precipitate PDCP particles. Particleswere pelleted at 11000 rpm for 30 minutes in 250 ml Oakridge tubes at 4°C. in a Sorvall RC5B centrifuge, then resuspended in 40 ml water and 8ml 2.5M NaCl/20% PEG 6000 added to reprecipitate particles, thenincubated on ice for 20 minutes. Particles were again pelleted at 11000rpm for 30 minutes in 50 ml Oakridge tubes at 4° C. in a Sorvall RC5Bcentrifuge, then resuspended in 5 ml PBS buffer, after removing alltraces of PEG/NaCl with a pipette. Bacterial debris was removed by a 5minute 13500 rpm spin in a microcentrifuge. The supernatant was filteredthrough a 0.45 μm polysulfone syringe filter, adjusted to 20% glyceroland stored at −70° C.

EXAMPLE 9 Isolation of Binding Activity from a N-Terminal Display PDCPLibrary of Human scFvs

The ability to select binding activities to a target of interest from ahuman antibody library is important due to the possibility of generatingtherapeutic human antibodies. In addition, such libraries allow theisolation of antibodies to targets which cannot be used for traditionalmethods of antibody generation due to toxicity, low immunogenicity orethical considerations. In this example we demonstrate the isolation ofspecific binding activities against a peptide antigen from a PDCPlibrary of scFvs from an un-immunised human.

The generation of the library, used for the isolation of bindingactivities in this example, is described in Example 8.

Substance P is an eleven amino acid neuropeptide involved ininflammatory and pain responses in vivo. It has also been implicated ina variety of disorders such as psoriasis and asthma amongst others(Misery, L. 1997, Br. J. Dertmatol., 137: 843-850; Maggi, C. A. 1997,Regul. Pept. 70: 75-90; Choi, D. C. & Kwon, O. J., 1998, Curr. Opin.Pulm. Med., 4: 16-24). Human antibodies which neutralise this peptidemay therefore have some therapeutic potential. As this peptide is toosmall to coat efficiently on a tube, as described in Example 3,selection of binding activities was performed in-solution, usingN-terminal biotinylated substance P and capturing bound PDCP particleson streptavidin-coated magnetic beads.

Enrichment for Substance P Binding PDCP Particles.

An aliquot of approximately 10¹³ a.r.u. IgM and IgD scFv library stockwas mixed with 1 μg biotinylated substance P in 800 μl 4% BSA/0.1% Tween20/PBS, and allowed to bind for two hours at ambient temperature. BoundPDCPs were then captured onto 1 ml of BSA blocked streptavidin coatedmagnetic beads for 10 minutes at ambient temperature. The beads werecaptured to the side of the tube with a magnet (Promega), and unboundmaterial discarded. The beads were washed eight times with 1 ml PBS/0.1%Tween 20/10 μg/ml streptavidin, then two times with 1 ml of PBS bymagnetic capture and removal of wash buffer. After the final wash boundPDCPs were eluted with 1 ml of freshly prepared 0.1M triethylamine for10 minutes, the beads were captured, and eluted particles transferred to0.5 ml 1M Tris-HCl pH 7.4. Neutralised particles were added to 10 ml logphase TG1 E. coli bacteria and incubated at 37° C. without shaking for30 minutes, then with shaking at 200 rpm for 30 minutes. 10⁻³, 10⁻⁴ &10⁻⁵ dilutions of the infected culture were prepared to estimate thenumber of particles recovered, and the remainder was spun at 4000 rpmfor 10 minutes, and the pellet resuspended in 300 μl 2×TY medium byvortex mixing. Bacteria were plated onto 2×TY agar plates supplementedwith 1% glucose and 100 g/ml ampicillin. Colonies were allowed to growovernight at 30° C. A 100-fold concentrated PDCP stock was prepared froma 200 ml amplified culture of these bacteria as described above, and 0.5ml used in as second round of selection with 500 ng biotinylatedsubstance P. For this round 100 μg/ml streptavidin was included in thewash buffer.

ELISA Identification of Binding Clones.

Binding clones were identified by ELISA of 96 individual PDCP culturesprepared as described in Example 3 from colonies recovered after thesecond round of selection. A Dynatech Immulon 4 ELISA plate was coatedwith 200 ng/well streptavidin in 100 μl/well PBS for 1 hour at 37° C.The plate was washed 3×200 μl/well PBS and incubated with long/wellbiotinylated substance P in 100 μl/well PBS for 30 minutes at 37° C. Theplate was washed 3×200 μl/well PBS and blocked for 1 hour at 37° C. with200 μl/well 2% Marvel non-fat milk powder/PBS and then washed 2×200μl/well PBS. 50 μl PDCP culture supernatant was added to each wellcontaining 50 μl/well 4% Marvel/PBS, and allowed to bind for 1 hour atambient temperature. The plate was washed three times with 200 μl/wellPBS/0.1% Tween 20, then three times with 200 μl/well PBS. Bound PDCPswere detected with 100 μl/well, 1:5000 diluted anti-M13-HRP conjugate(Pharmacia) in 2% Marvel/PBS for 1 hour at ambient temperature and theplate washed six times as above. The plate was developed for 10 minutesat ambient temperature with 100 μl/well freshly prepared TMB(3,3′,5,5′-Tetramethylbenzidine) substrate buffer (0.005% H₂O₂, 0.1mg/ml TMB in 24 mM citric acid/52 mM sodium phosphate buffer pH 5.2).The reaction was stopped with 100 μl/well 12.5% H₂SO₄ and read at 450nm. Out of 96 clones tested, 10 gave signals greater than twicebackground (background=0.05).

Characterization of a Binding Clone.

A 50-fold concentrated PDCP stock was prepared from a 100 ml amplifiedculture of a single ELISA positive clone as described above. 10 μl perwell of this stock was tested in ELISA as described above for binding tostreptavidin, streptavidin-biotinylated-substance P andstreptavidin-biotinylated-CGRP (N-terminal biotinylated). Binding wasonly observed in streptavidin-biotinylated-substance P coated wellsindicating that binding was specific. In addition, binding tostreptavidin-biotinylated substance P was completely inhibited byincubating the PDCP with 1 μg/ml free substance P (see FIG. 8). The scFvVH (SEQ ID Nos 15 and 16) and VL (SEQ ID Nos 17 and 18) DNA and aminoacid sequence was determined by DNA sequencing with oligonucleotidesM13REV (SEQ ID No27) and ORSEQFOR (SEQ ID No 36) and is shown in FIG. 9.

The results indicate that target binding activities can be isolated fromPDCP display libraries of human scFv fragments.

EXAMPLE 10

In another example the invention provides methods for screening a DNAlibrary whose members require more than one chain for activity, asrequired by, for example, antibody Fab fragments for ligand binding. Toincrease the affinity of an antibody of known heavy and light chainsequence, libraries of unknown light chains co-expressed with a knownheavy chain are screened for higher affinity antibodies. The known heavychain antibody DNA sequence is joined to a nucleotide sequence encodinga oestrogen receptor DNA binding domain in a phage vector which does notcontain the oestrogen receptor HRE sequence. The antibody DNA sequencefor the known heavy chain (VH and CH1) gene is inserted in the 5′ regionof the oestrogen receptor DBD DNA, behind an appropriate promoter andtranslation sequences and a sequence encoding a signal peptide leaderdirecting transport of the downstream fusion protein to the periplasmicspace. The library of unknown light-chains (VL and CL) is expressedseparately from a phagemid expression vector which also contains theoestrogen receptor HRE sequence. Thus when both heavy and light chainsare expressed in the same host cell, following infection with the phagecontaining the heavy chain-DBD fusion, the light chain phagemid vectoris preferentially packaged into mature phage particles as singlestranded DNA, which is bound by the heavy chain-DBD fusion proteinduring the packaging process. The light chain proteins are transportedto the periplasm where they assemble with the heavy chain that is fusedto the DBD protein as it exits the cell on the PDCP. In this example theDBD fusion protein and the HRE DNA sequences are not encoded on the samevector, the unknown peptide sequences are present on the same vector asthe HRE sequence. Peptide display carrier packages (PDCP) which encodethe protein of interest can then be selected by means of a ligandspecific for the antibody.

TABLE 1 (i) Oligonucleotide primers used for human scFv libraryconstruction cDNA synthesis primers IgMCDNAFORTGGAAGAGGCACGTTCTTTTCTTT         (SEQ ID 38) IgDCDNAFORCTCCTTCTTACTCTTGCTGGCGGT        (SEQ ID 37) IgκCDNAFORAGACTCTCCCCTGTTGAAGCTCTT        (SEQ ID 39) IgλCDNAFORTGAAGATTCTGTAGGGGCCACTGTCTT     (SEQ ID 40) JHFOR primers JH1-2FORTGAACCGCCTCCACCTGAGGAGACGGTGACCAGGGTGCC   (SEQ ID 41) JH3FORTGAACCGCCTCCACCTGAAGAGACGGTGACCATTGTCCC   (SEQ ID 42) JH4-5FORTGAACCGCCTCCACCTGAGGAGACGGTGACCAGGGTTCC   (SEQ ID 43) JH6FORTGAACCGCCTCCACCTGAGGAGACGGTGACCGTGGTCCC   (SEQ ID 44)VH familyBAKprimers VH1BAKTTTTTGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTCTGG   (SEQ ID 45) VH2BAKTTTTTGGCCCAGCCGGCCATGGCCCAGGTCAACTTAAGGGAGTCTGG   (SEQ ID 46) VH3BAKTTTTTGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTCTGG   (SEQ ID 47) VH4BAKTTTTTGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCGGG   (SEQ ID 48) VH5BAKTTTTTGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGTTGCAGTCTGC   (SEQ ID 49) VH6BAKTTTTTGGCCCAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGG   (SEQ ID 50)Light chain FOR primers SCFVKFORTTATTCGCGGCCGCCTAAACAGAGGCAGTTCCAGATTTC       (SEQ ID 51) SCFVλFORGTCACTTGCGGCCGCCTACAGTGTGGCCTTGTTGGCTTG        (SEQ ID 52)VK family BAK primers VK1BAKTCTGGCGGTGGCGGATCGGACATCCAGATGACCCAGTCTCC     (SEQ ID 53) VK2BAKTCTGGCGGTGGCGGATCGGATGTTGTGATGACTCAGTCTCC     (SEQ ID 54) VK3BAKTCTGGCGGTGGCGGATCGGAAATTGTGTTGACGCAGTCTCC     (SEQ ID 55) VK4BAKTCTGGCGGTGGCGGATCGGACATCGTGATGACCCAGTCTCC     (SEQ ID 56) VK5BAKTCTGGCGGTGGCGGATCGGAAACGACACTCACGCAGTCTCC     (SEQ ID 57) VK6BAKTCTGGCGGTGGCGGATCGGAAATTGTGCTGACTCAGTCTCC     (SEQ ID 58) JK FOR primersJK1FOR TTCTCGTGCGGCCGCCTAACGTTTGATTTCCACCTTGGTCCC    (SEQ ID 59) JK2FORTTCTCGTGCGGCCGCCTAACGTTTGATCTCCAGCTTGGTCCC    (SEQ ID 60) JK3FORTTCTCGTGCGGCCGCCTAACGTTTGATATCCACTTTGGTCCC    (SEQ ID 61) JK4FORTTCTCGTGCGGCCGCCTAACGTTTGATCTCCACCTTGGTCCC    (SEQ ID 62) JK5FORTTCTCGTGCGGCCGCCTAACGTTTAATCTCCAGTCGTGTCCC    (SEQ ID 63) Vλfamily BAK primers Vλ1BAK TCTGGCGGTGGCGGATCGCAGTCTGTGTTGACGCAGCCGCC    (SEQ ID 64) Vλ2BAK TCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCTGC    (SEQ ID 65) (ii) Oligonucleotide primers used for human scFv libraryconstruction Vλ3aBAK TCTGGCGGTGGCGGATCGTCCTATGTGCTGACTCAGCCACC    (SEQ ID 66) Vλ3bBAK TCTGGCGGTGGCGGATCGTCTTCTGAGCTGACTCAGGACCC    (SEQ ID 67) Vλ4BAK TCTGGCGGTGGCGGATCGCACGTTATACTGACTCAACCGCC    (SEQ ID 68) Vλ5BAK TCTGGCGGTGGCGGATCGCAGGCTGTGCTCACTCAGCCGTC    (SEQ ID 69) Vλ6BAK TCTGGCGGTGGCGGATCGAATTTTATGCTGACTCAGCCCCA    (SEQ ID 70) Jλ primers Jλ1FORTTCTCGTGCGGCCGCCTAACCTAGGACGGTGACCTTGGTCCC     (SEQ ID 71) Jλ2-3FORTTCTCGTGCGGCCGCCTAACCTAGGACGGTCAGCTTGGTCCC     (SEQ ID 72) Jλ4-5FORTTCTCGTGCGGCCGCCTAACCTAAAACGGTGAGCTGGGTCCC     (SEQ ID 73)Linker primers LINKAMP3 CGATCCGCCACCGCCAGA      (SEQ ID 74) LINKAMP5GTCTCCTCAGGTGGAGGC       (SEQ ID 75) LINKAMP3TCGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGAC   (SEQ ID 76)

The invention claimed is:
 1. A peptide display carrier package (PDCP),said package comprising a recombinant polynucleotide-chimeric proteincomplex wherein the chimeric protein has a nucleotide binding portionand a target peptide portion, wherein said recombinant polynucleotidecomprises a sequence encoding said chimeric protein and a nucleotidesequence motif which is specifically bound by said nucleotide bindingportion of said chimeric protein, wherein said nucleotide bindingportion comprises a DNA binding domain of an oestrogen or progesteronereceptor, and wherein at least the chimeric protein-encoding portion ofthe recombinant polynucleotide not bound by the chimeric proteinnucleotide binding portion is protected by a viral coat protein.
 2. Apeptide display carrier package (PDCP) as claimed in claim 1, whereinsaid target peptide portion is displayed externally on the package.
 3. Apeptide display carrier package (PDCP) as claimed in claim 2 whereinsaid recombinant polynucleotide includes a linker sequence between thenucleotide sequence encoding the nucleotide binding portion and thenucleotide sequence encoding the target peptide portion.
 4. A peptidedisplay carrier package (PDCP) as claimed in claim 3 wherein saidrecombinant polynucleotide has two or more nucleotide sequence motifseach of which can be bound by the nucleotide binding portion of thechimeric protein.
 5. A peptide display carrier package (PDCP) as claimedin claim 4 wherein said recombinant polynucleotide is bound to saidchimeric protein as single stranded DNA.
 6. A peptide display carrierpackage (PDCP) as claimed in claim 5 wherein said target peptide portionis located at the N and/or C terminal of the chimeric protein.
 7. Apeptide display carrier package (PDCP) as claimed in claim 6 which isproduced in a host cell transformed with said recombinant polynucleotideand extruded therefrom without lysis of the host cell.